During human and mouse embryogenesis, primary hematopoietic cells are generated in the yolk sac and para-aortic splanchnopleura/aorta-genital ridges-mesonephros (AGM) region; however, only cells generated in the AGM region are believed to contribute to hematopoietic stem cells (Godin I, et al., Nat Rev Immunol. 2002; 2:593-604). Both extra- and intra-embryonic hematopoietic cells develop in close association with endothelial cells from common hematoendothelial precursors, which were identified within early embryonic and embryonic stem cell (ESC)-derived cell populations expressing endothelial markers (VEGF-R2 (KDR), VE-cadherin, CD31, Tie2) (Tavian M, et al. Ann N Y Acad. Sci. 2005; 1044:41-50; Li W, et al., Stem Cells Dev. 2005; 14:44-54; Jaffredo T, et al., Int J Dev Biol. 2005; 49:269-277; Fraser S T, et al., Exp Hematol. 2002; 30:1070-1078; Wang L, et al., Immunity, 2004; 21:31-41; Nishikawa S I, et al., Development, 1998; 125:1747-1757; Choi K, et al., Development 1998; 125:725-732). These precursors are of particular interest for studies on the divergence of endothelial and hematopoietic cell lineages and establishment of hematopoietic stem cells, however, their identification requires a reliable separation of the earliest lineage-committed progeny, since hematopoietic and endothelial derivatives may share a common phenotype at early stages of development and still may not express typical lineage-specific markers. For example, in the mouse embryo, CD45, the most specific marker of hematopoietic lineage, is not expressed on the earliest hematopoietic progenitors arising in the yolk sac and AGM region; however, these progenitors can be identified by expression of CD41 molecule, which is specific for megakaryocytic lineage in adults (Li W, et al., Stem Cells Dev. 2005; 14:44-54; Mikkola H K, et al., Blood 2003; 101:508-516; Bertrand J Y, et al., Proc Natl Acad Sci USA, 2005; 102:134-139; Ferkowicz M J, et al., Development 2003; 130:4393-4403; Mitjavila-Garcia M T, et al., Development 2002; 129:2003-2013).
Hematopoietic differentiation of human ESCs (hESCs) reproduces many aspects of embryonic hematopoiesis and provides an in vitro model to elucidate mechanisms of early hematopoietic commitment, (Keller G., Genes Dev. 2005; 19:1129-1155; Wang L, et al., Exp Hematol. 2005; 33:987-996) practically inaccessible in the human embryo (Tavian M, et al., Int J Dev Biol. 2005; 49:243-250). Several hESC hematopoietic differentiation systems based on either coculture with stromal cells (Kaufman D S, et al., Proceedings of the National Academy of Sciences of the United States of America. 2001; 98:10716-10721; Qiu C, et al., Exp Hematol. 2005; 33:1450-1458; Vodyanik M A, et al., Blood 2005; 105:617-626) or formation of embryoid bodies (Chadwick K, et al., Blood 2003; 102:906-915; Zambidis E T, et al., Blood 2005; 106:860-870; Ng E S, et al., Blood 2005; 106:1601-1603) have been established. Recently, we described hESC differentiation in coculture with OP9 stromal cells that resulted in a highly efficient generation of hematopoietic progenitors after 4-5 days of coculture (Vodyanik M A, et al., Blood 2005; 105:617-626). We and others have demonstrated that hematopoietic clonogenic progenitors arise within CD34+ cell population before emergence of CD45+ cells, suggesting that the first hematopoietic progenitors in humans, like in mice, can not be identified by CD45 expression (Kaufman D S, et al., Proceedings of the National Academy of Sciences of the United States of America 2001; 98:10716-10721; Vodyanik M A, et al., Blood 2005; 105:617-626; Zambidis E T, et al., Blood 2005; 106:860-870; Ng E S, et al., Blood 2005; 106:1601-1603).